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Vol. 14, Issue 6, 2342-2356, June 2003
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* Department of Cell Biology, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205;
Division of Biology, Section of Cell and Developmental Biology, University of
California, San Diego, La Jolla, California 92093
Submitted December 4, 2002;
Revised January 21, 2003;
Accepted January 30, 2003
Monitoring Editor: Thomas D. Fox
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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;
Bereiter-Hahn and Voth, 1994).
In the yeast S. cerevisiae, mitochondrial fusion and division are
highly regulated during growth, mating, and sporulation and control the
structure and function of this essential organelle
(Hermann and Shaw, 1998;
Yaffe, 1999;
Jensen et al., 2000;
Shaw and Nunnari, 2002).
Mitochondrial fusion in yeast requires the Fzo1 protein
(Hermann et al.,
1998; Rapaport et
al., 1998; Sesaki and
Jensen, 1999) and the Ugo1 protein
(Sesaki and Jensen, 2001).
Fzo1p is a homolog of Drosophila fuzzy onions protein, which is
required for mitochondrial fusion during fly spermato-genesis
(Hales and Fuller, 1997).
Fzo1p is located in the mitochondrial outer membrane (OM) with an N-terminal
GTPase domain facing the cytosol (Hermann
et al., 1998;
Rapaport et al.,
1998). Ugo1p is also an OM protein with a single transmembrane
domain (Sesaki and Jensen,
2001). Cells disrupted for FZO1 or UGO1 contain
many small mitochondrial fragments instead of the few tubular mitochondria
seen in wild-type cells (Hermann et
al., 1998; Rapaport
et al., 1998; Sesaki
and Jensen, 2001). Defects in mitochondrial fusion in these
mutants have been directly demonstrated using a mating assay. In addition to
fusion, Fzo1p and Ugo1p are also important for mitochondrial maintenance of
mitochondrial DNA (mtDNA). fzo1 and ugo1 mutants lose mtDNA,
but the mechanism by which mtDNA is lost in these mutants is not understood
and appears to be a secondary consequence of mitochondrial morphology defect
(Bleazard et al.,
1999; Sesaki and Jensen,
1999)
The fragmentation of mitochondria in fzo1 and ugo1
mutants depends on mitochondrial division
(Bleazard et al.,
1999; Sesaki and Jensen,
1999,
2001). Mitochondrial division
is mediated by Dnm1p, a dynamin-related GTPase
(Gammie et al., 1995;
Bleazard et al., 1999;
Sesaki and Jensen, 1999).
Cells disrupted for DNM1 contain a single mitochondrion consisting of
a network of interconnected tubules. fzo1 dnm1 and ugo1 dnm1
double mutants contain nearly wild-type, tubular-shaped mitochondria,
suggesting that mitochondrial shape and number are normally regulated by a
balance between division and fusion (Sesaki and Jensen,
1999,
2001). Like mitochondrial
fragmentation, the loss of mtDNA in fzo1 and ugo1 mutants
requires Dnm1p, and fzo1 dnm1, and ugo1 dnm1 double mutants
maintain mtDNA (Bleazard et al.,
1999; Sesaki and Jensen,
1999,
2001).
Taking advantage of the mtDNA phenotype of fzo1 mutants, we
developed a genetic screen for mutants defective in mitochondrial fusion,
which yielded ugo1 mutants
(Sesaki and Jensen, 2001).
Using this screen, we also identified five mgm1 mutants, leading us
to investigate the role of Mgm1p in mitochondrial fusion. mgm1 was
originally identified as a mutant that was unable to maintain mtDNA
(Jones and Fangman, 1992).
Later studies show that mgm1 mutants also lose normal mitochondrial
morphology and contain mitochondrial fragments, which often aggregate into
clusters within the yeast cells (Guan
et al., 1993; Shepard
and Yaffe, 1999). These clumped mitochondria are not efficiently
inherited from mother to daughter cells
(Shepard and Yaffe, 1999).
Mgm1p contains a predicted GTP-binding motif that is homologous to the GTPase
dynamin (Jones and Fangman,
1992), and this GTPase domain is essential for the function of
Mgm1p (Shepard and Yaffe,
1999). There are two species of Mgm1p, a 90- and a 100-kDa form,
but the relationship between these two forms is unknown
(Shepard and Yaffe, 1999).
Mgm1p has been localized to mitochondria, but its submitochondrial location is
controversial. Although Shepard and Yaffe
(1999) localized Mgm1p to the
OM, Wong et al.
(2000) found Mgm1p in the
intermembrane space, peripherally associating with the inner membrane (IM).
Therefore, the actual location of Mgm1p remains unclear.
Because the phenotypes of mgm1 mutants are similar to those of
mutants defective in mitochondrial fusion, such as fzo1
(Hermann et al.,
1998; Rapaport et
al., 1998) and ugo1
(Sesaki and Jensen, 2001), the
ability to fuse mitochondria was examined in mgm1 and mgm1
dnm1 mutants (Wong et al.,
2000). Supporting Mgm1p's involvement in mitochondrial fusion,
temperature-sensitive mgm1-5 mutants were unable to fuse mitochondria
during yeast mating. However, mgm1-5 dnm1 double mutants fused their
mitochondria at the restrictive temperature
(Wong et al., 2000).
Therefore, it is not clear whether Mgm1p is involved in mitochondrial fusion,
or in mitochondrial shape. In this article, using complete disruptions of the
MGM1 open reading frame, we show that both mgm1
and
mgm1 dnm1 cells are defective in mitochondrial
fusion, demonstrating that Mgm1p is essential for mitochondrial fusion. We
further show that the Mgm1p is required for both OM and IM fusion. Consistent
with its essential role in OM fusion, the majority of Mgm1p is associated with
the OM and physically interacts with Fzo1p and Ugo1p.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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).
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Plasmids
pSS1, a CEN-TRP1 plasmid expressing OM45-GFP was
constructed as follows. pKC2, a CEN-LEU2 plasmid carrying
OM45-GFP (Cerveny et al.,
2001) was digested with PvuI. The OM45-GFP fragment and
XhoI/NotI-digested pRS314
(Sikorski and Hieter, 1989)
were transformed into yeast and pSS1 was formed by homologous recombination
(Oldenburg et al.,
1997).
pHS70, a CEN-URA3 plasmid expressing CFP fused to the C termini of
Yta10p under the control of the GAL1 promoter was constructed as
follows. YTA10 was PCR amplified from yeast genomic DNA
(Hoffman and Winston, 1987)
using oligos 720 (5'-GGGCTCGAGATGATGATGTGGCAACG-3') and 721
(5'-GGGTCTAGAATTTGTTGCTGCAGGTG-3'). The PCR fragment was digested
with XhoI and XbaI and subcloned into
XhoI/XbaI-digested pGAL1-COX4-CFP (HS52;
Sesaki and Jensen, 2001).
pHS71, a CEN-URA3 plasmid expressing YFP fused to the C termini of Yta10p under the control of the GAL1 promoter was constructed as follows. YFP was PCR amplified from pEYFP (Clontech Laboratories, Inc.) by using oligos 355 (5'-TGCTCTAGAATGGTGAGCA AGGGC-3') and 498 (5'-CCGGATCCTTACTTGTACAGCTCGTC-3'). The PCR fragment was digested with XbaI and BamHI and cloned into XbaI/BamHI-digested pHS70.
pHS72, a CEN-URA3 plasmid expressing YFP fused to the first 40
amino acids of Tom72p under the control of the GAL1 promoter was
constructed as follows. TOM72 was PCR amplified from yeast genomic DNA using
oligos 549 (5'-GGGCTCGAGATGGCCGAAAA CTCCCTC-3') and 548
(5'-GAAGCGGCCGCCCTGCTGCTTAATTTGCTG-3'). The PCR fragment was
digested with XhoI and NotI and cloned into
XhoI/NotI-digested pRS316GU
(Nigro et al., 1992),
forming pHS72–1. YFP was PCR amplified from pEYFP (Clontech
Laboratories, Inc., Palo Alto, CA) by using oligos 347
(5'-AAGGAAAAAAGCGGCCGCATGGTGAGCA AGGGC-3') and 498. The PCR
fragment was digested with NotI and BamHI and cloned into
NotI/BamHI-digested pHS72–1, forming pHS72.
pHS77, a CEN-TRP1 plasmid expressing His10-HA-Fzo1p was
constructed as follows. The promoter of FZO1 was PCR amplified from
yeast genomic DNA using oligos 904
(5'-GGGCTCGAGTGTTCCACTTTCTTGCG-3') and 905
(5'-GGGGAATTCCGTTAAATGAGCCTACC-3'). The PCR fragment was digested
with XhoI and EcoRI and cloned into
XhoI/EcoRI-digested pRS314
(Sikorski and Hieter, 1989),
forming pRS314-Fp. The HA epitope was PCR amplified from pGTEP
(Tyers et al., 1992)
using oligos OSM373
(5'-TTGAATTCATGCACCACCACCACCACCACCACCACCACCCTACCCATACGATGTTCCTG-3')
and 906 (5'-GGGGCGGCCGCGAGCAGCGTAATCTGGAACG-3). The PCR fragment was
digested by EcoRI and NotI, and cloned into
EcoRI/NotI-digested pRS314-Fp, forming pRS314-Fp- HA. The
open reading frame of FZO1 was PCR amplified from yeast genomic DNA
using oligos 907 (5'-GGGGCGGCCGCTCTGAAGGAAAACAACAATTC-3') and 908
(5'-GGGCCGCGGAAAATGGACCTGCTTGG-3'). The PCR fragment was digested
with NotI and SacII, and cloned into
NotI/SacII-digested pRS314-Fp-HA, forming pHS77.
pHS73, a TRP1 plasmid expressing Mgm1p, was constructed as
follows. MGM1 was PCR amplified from yeast genomic DNA using oligos
758 (5'-GGGCTCGAGGTGTCAGTAAATAACAGAG-3') and 711
(5'-AATGCGGCCGCCTTAGATGAAGGGTATG-3'). The PCR fragment was
digested with XhoI and NotI and cloned into
XhoI/NotI-digested pRS304
(Sikorski and Hieter,
1989).
To introduce mutations in the GTPase domain of MGM on pHS73, site-directed mutagenesis (QuickChange, Stratagene, La Jolla, CA) was performed using the following mutagenic oligonucleotides 804 (5'-GGTTCACAATCGTCTGGTGCATCCTCAGTACTAGAATCC-3') and 805 (5'-GGATTCTAGTACTGAGGATGCACCAGACGATTGTGA ACC-3') for pHS74 carrying mgm1K223A, 806 (5'-CACAATCGTCTGGTAAAAACTCAGTACTAGAATCCATTG-3') and 807 (5'-CAATGGATTCTAGTACTGAGTTTTTACCAGACGATTGTG-3') for pHS75 carrying mgm1S224N, and 808 (5'-GGTTCCAACATGGTCGCAAGAAGACCCATTGAATTG-3') and 809 (5'-CAATTCAATGGGTCTTCTTGCGACCATGTTGGAACC-3') for pHS76 carrying mgm1T244A. The mutations were confirmed by DNA sequencing.
Gene Disruption
Complete disruptions of the MGM1 and ATP21genes were
constructed by PCR-mediated gene replacement as described (Lawrence, 1991)
into diploid strain FY833/844 (Winston
et al., 1995). For mgm1::kanMX4, we used the
kanMX4 gene from the pRS400 plasmid (Brachmann, 1998). Heterozygous
diploids were sporulated and dissected to obtain MATa
mgm1 strain YRJ1383 and MAT
mgm1 strain
YRJ1384. MATa mgm1 dnm1 strain
YRJ1398 and MAT mgm1 dnm1 strain
YRJ1493 were constructed by crossing MATa mgm1 strain
YRJ1383 and MAT dnm1 strain YRJ1290
(Sesaki and Jensen, 2001). For
atp21::HIS3 and mmm1::HIS3, the HIS3 gene from the
pRS303 plasmid (Sikorski and Hieter,
1989) was used. Heterozygous diploids were sporulated and
dissected to obtain MATa atp21 strain YRJ1564 and
MAT atp21 strain YRJ1565. To create MATa
mmm1 strain YRJ1355 and MAT mmm1 strain
YRJ1356, the HIS3 gene from the pRS303 plasmid
(Sikorski and Hieter, 1989)
was used.
Chromosomal Integration of mgm1 GTPase
Mutations
To create MGM1 dnm1, mgm1K223 dnm1,
mgm1S224N dnm1, and mgm1T244A
dnm1 cells, pHS73, pHS74, pHS75, and pHS76 were digested with
NdeI and transformed into MATa mgm1
dnm1 strain YRJ1398 and MAT mgm1
dnm1 strain YRJ1493. Integration of these plasmids into
chromosomes was confirmed by PCR.
Microscopy
Cells were observed using a Zeiss Axioskop microscope (Thornwood, NY) with
a 100x Plan-Neofluar objective. Fluorescence and differential
interference contrast images were captured with a Hamamatsu Orca ER using Open
Lab software version 3.0.8 (Improvision Inc., Lexington, MA).
Electron microscopy was performed as previously described (Reider et al., 1996). Briefly, cells were fixed in 3% glutaraldehyde and embedded in Spurrr's resin, and thin sections were cut on a Reichert Ultracut T ultramicrotome (Leica, Deerfield, IL). Samples were examined using a Philips EM420 electron microscope (FEI Co., Peabody, MA).
Mitochondrial Fusion Assay
Mitochondrial fusion during mating was observed as described
(Azpiroz and Butow, 1995;
Nunnari et al., 1997;
Okamoto et al.,
1998). MATa strains carrying pGAL1-YTA10-CFP (pHS70) and
MAT strains carrying pGAL1-YTA10-YFP (pHS71) were grown to log
phase in SGalSuc medium overnight, pelleted by centrifugation, washed, and
resuspended in 1 ml of YEPD medium. Same OD600 units of
MATa and MAT cells were mixed and collected by
centrifugation. Cells were resuspended in YEPD medium at 0.025
OD600 units/µl and placed on a nitrocellulose membrane at 1.2
OD600 units/cm2. Excess solution was removed by placing
the membrane on filter paper. The nitrocellulose membrane was then incubated
on YEPD medium, adjusted to pH 4.5 using citric acid
(Azpiroz and Butow, 1995), at
30°C for 3 h. Zygotes were examined by fluorescence microscopy.
For fusion of the mitochondrial OM, MAT strains carrying
pGAL1-TOM72-YFP (pHS72) were grown to log phase in SRafSuc medium overnight,
pelleted by centrifugation, and resuspended in SGalSuc medium to an
OD600 of 0.2 for 2 h to induce TOM72-YFP expression. MATa
strains carrying pGAL1-YTA10-CFP (pHS70) were grown to log phase in SGalSuc
medium overnight. Cells were mated as described above.
Submitochondrial Fractionation
Mitochondria were isolated from wild-type cells (FY833) as previously
described (Daum et al.,
1982). Separation of OM and IM vesicles on sucrose gradients was
performed as described (Ryan et
al., 1994). For protease digestions, we resuspended 100 µg
of mitochondria in 1 ml of 250 mM sucrose, 20 mM HEPES-KOH, pH 7.4.
Mitochondria were treated with either 50 µg/ml trypsin for 20 min on ice,
followed by the addition of 200 µg/ml soybean trypsin inhibitor, or 50
µg/ml proteinase K, followed by the addition of 1 mM
phenylmethylsulfonylfluoride. To disrupt the mitochondrial OM, 100 µg of
mitochondria were incubated in 1 ml of 20 mM HEPES-HCl, pH 7.4, for 40 min on
ice.
Proteins were separated by SDS-PAGE
(Laemmli, 1970) and
transferred to Immobilon filters (Haid and Suissa, 1983). Filters were probed
with antibodies to Mgm1p (Shepard and
Yaffe, 1999), The 
subunit of F1-ATPase, Tim23p
(Emtage and Jensen, 1993),
Om45p (Yaffe et al.,
1989), Cox4p, and Mas2p (Jensen and Yaffe, 1988), all at 1:10,000
dilutions, or Tom37p (Gratzer et
al., 1995) and Aac2p (a gift from N. Pfanner, Institut fur
Biochemie and Molekularbiologie, Universitat Freiburg, Germany) at 1:5000
dilutions. Immune complexes were visualized using 1:10,000 dilution of
HRP-conjugated secondary antibodies (Amersham Pharmacia Biotech, Piscataway,
NJ) followed by chemiluminescence (SuperSignal; Pierce Chemical Co., Rockford,
IL).
Immune Precipitation
Mitochondria were isolated from strain YRJ1282 expressing HA-Fzo1p from
pHS77 and strain YRJ1294 expressing myc-Ugo1p from pHS57
(Sesaki and Jensen, 2001), as
described before (Daum et al.,
1982). For immune precipitations, 0.5 mg of mitochondria was
solubilized in 0.5 ml of IP buffer (1% Triton X-100, 50 mM NaCl, 30 mM
HEPES-KOH, pH 7.4) containing protease inhibitors (1 µg/ml aprotinin, 1
µg/ml leupeptin, 1 mM phenylmethylsulfonylfluoride, 10 µM
trans-epoxy-succinyl-L-leucylamido(4-guanidino)butane, 1 µg/ml chymostatin,
1 µg/ml pepstatin A) at 4°C with gentle agitation for 10 min. After
centrifugation at 12,500 x g for 10 min, 100 µl of 50%
slurry of antibody-coupled beads was added to the supernatant. We coupled the
HA antibody (12CA5; Niman et al.,
1983) or the myc antibody (9E10;
Evan et al., 1985) to
beads using Seize Immunoprecipitation Kit (Pierce) according to manufacturer's
instructions. Samples were incubated at 4°C with gentle agitation for 6 h.
Beads were washed three times with 0.4 ml of wash buffer (0.1% Triton X-100,
50 mM NaCl, 30 mM HEPES-KOH, pH 7.4) containing protease inhibitors. The bound
proteins were eluted by 200 µl of 2x sample buffer (2% SDS, 20%
glycerol, 10 µg/ml bromophenol blue, 200 mM Tris-HCl, pH 6.8) at 60°C
for 5 min. We added 8 µl of 14.4 M -mercaptoethanol to the eluates
and boiled for 5 min. Proteins were separated by SDS-PAGE
(Laemmli, 1970) and analyzed
by immune blotting with antibodies to the myc epitope (PRB-150; Covance,
Berkeley, CA), the HA epitope (12CA5;
Niman et al., 1983),
Mgm1p, OM45p, and Tim23p, all at 1:10,000 dilutions.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). In
addition to fzo1 and ugo1 mutants, we also isolated 5
mgm1 mutants. To investigate whether Mgm1p functions in mitochondrial
fusion, we disrupted MGM1, DNM1, and both MGM1 and
DNM1 in yeast cells, and examined mitochondria and mtDNA in these
mutant cells. Mitochondria were visualized using an OM-targeted GFP fusion
protein, OM45-GFP (Cerveny et al.,
2001). Wild-type cells contained 5–10 mitochondrial tubules
with occasional branches. In contrast, mgm1 cells showed many
small mitochondrial fragments, which often aggregated in the cell
(Figure 1A). The mitochondria
seen in mgm1 cells are similar to those seen in
fzo1 and ugo1 cells
(Hermann et al.,
1998; Rapaport et
al., 1998; Sesaki and
Jensen, 2001). The fragmentation of mitochondria in
fzo1 and ugo1 cells has been shown to result
from a block in fusion along with continuing mitochondrial division. In
dnm1 cells mitochondria form a single network consisting of
interconnected tubules, due to ongoing fusion in the absence of mitochondrial
division (Figure 1A;
Bleazard et al., 1999;
Sesaki and Jensen, 1999). In
contrast, in the majority of mgm1 dnm1 cells
(88%, n = 100), mitochondria were found to form elongated tubules with some
branches, similar to those seen in wild-type cells. As seen in
fzo1 dnm1 and ugo1
dnm1 cells (Sesaki and Jensen,
1999,
2001), these mitochondrial
tubules were often collapsed to one side of the cell and appeared to be
bundled.
|
The loss of mtDNA in mgm1 cells was also rescued by
disruption of DNM1. Wild-type and mutant cells were stained by
4',6'-diamidino-2-phenylindole (DAPI) and tested for growth on
nonfermentable carbon sources. We found that mgm1 cells lack
mtDNA nucleoids (Figure 1A) and
fail to grow on glycerol and ethanol-containing medium (YEPGE;
Figure 1B). However, wild-type,
dnm1 and mgm1 dnm1 cells all
contained similar amounts of mtDNA nucleoids. Consistent with previous studies
(Wong et al., 2000;
Fekkes et al., 2000),
mgm1 dnm1 cells were able to grow on YEPGE
medium, demonstrating that the double mutants contain mtDNA. We note that
mgm1 dnm1 cells, like fzo1
dnm1 and ugo1 dnm1 cells, grew
more slowly than wild-type and dnm1 cells on both YEPD and
YEPGE media. This growth appears to be due to lack of Mgm1p because
mgm1 dnm1 cells contain normal amounts of
mtDNA. Our results demonstrate that disruption of MGM1 causes
fragmentation of mitochondria and loss of mtDNA in a Dnm1p-dependent manner.
Because the phenotypes seen in mgm1 cells are almost identical
to those seen in fzo1 and ugo1 mutants, which
are defective in mitochondrial fusion
(Hermann et al.,
1998; Rapaport et
al., 1998; Sesaki and
Jensen, 2001), our data raise the possibility that Mgm1p likewise
functions in mitochondrial fusion.
mgm1 and mgm1 dnm1 Cells Are
Defective in Mitochondrial Fusion
To determine directly whether Mgm1p is required for mitochondrial fusion,
we examined the ability of mgm1 and mgm1
dnm1 mutants to fuse their mitochondria during yeast cell
mating (Azpiroz and Butow,
1995; Nunnari et al.,
1997; Okamoto et al.,
1998; Sesaki and Jensen,
1999,
2001). The mitochondria in
MATa cells were marked using an IM-targeted cyan fluorescent protein
(YTA10-CFP) expressed from pHS70. In MAT cells, mitochondria
were labeled by an IM-targeted yellow fluorescent protein (YTA10-YFP) carried
on pHS71. Both plasmids express the fusion protein under the control of the
inducible GAL1 promoter. MATa and MAT cells
were pregrown in galactose-containing medium to induce the expression of the
fusion proteins and transferred to glucose-containing medium to inhibit their
further synthesis. Cells were mixed and mated on glucose-containing medium. If
mitochondria fuse in the resulting zygotes, CFP and YFP fluorescence overlap
because of the diffusion of the YTA10-CFP and YTA10-YFP proteins in the IM. If
mitochondria fail to fuse, CFP and YFP are seen only in separate
organelles.
We found that mgm1 and mgm1
dnm1 cells fail to fuse their mitochondria.
Figure 2 shows representative
examples of zygotes containing a medial diploid bud from each mating mixture.
Fifty zygotes were examined for wild-type and each mutant. When two wild-type
haploids mated, mitochondria in the zygote fused and mixed their IM contents,
and the CFP and YFP fluorescence therefore overlapped. Mitochondrial fusion
also occurred in zygotes formed between dnm1 mutants. In
contrast, when two mgm1 cells were mated, mitochondrial
fragments containing only CFP or YFP were observed. Although the disruption of
DNM1 suppresses the fragmentation of mitochondria and the loss of
mtDNA, mgm1 dnm1 double mutants were still
unable to fuse their mitochondria. mgm1 dnm1
cells formed tubular mitochondria, but these tubules contained only CFP or
YFP. In mgm1 dnm1 zygotes, we frequently found
that two mitochondrial tubules, each derived from one parent, entered the
diploid bud and were closely positioned in the middle of zygotes, but
nonetheless these mitochondria did not fuse. Our results indicate that,
whether mitochondria exist as fragments or tubules, cells lacking Mgm1p are
defective in mitochondrial fusion.
|
Our findings were unexpected because a previous study showed that
mitochondria fuse normally in mgm1-5 dnm1 zygotes
(Wong et al., 2000).
mgm1-5 is a temperature-sensitive allele for MGM1. In a
mating assay, mitochondria failed to fuse in mgm1-5 cells at the
restrictive temperature, but mitochondria fusion was rescued in mgm1-5
dnm1 double mutants (Wong
et al., 2000). To test if the difference between our
results and those from previous studies results from the use of different
yeast strains, we examined mitochondrial fusion in the W303 strain used in the
previous study (Wong et al.,
2000). When a null allele of MGM1, mgm1, was
constructed in W303, we found mitochondrial fusion to be defective. This
fusion defect was seen in both mgm1 (our unpublished results)
and mgm1 dnm1 cells
(Figure 2). These results
clearly demonstrate that Mgm1p is required for mitochondrial fusion in two
different backgrounds.
Mutations in the GTPase Domain of Mgm1p Block Mitochondrial
Fusion
Mgm1p is a member of a family of proteins related to the dynamin GTPase.
The MGM1 gene carrying a mutation in its GTPase domain is unable to
rescue fragmentation of mitochondria and loss of mtDNA in
temperature-sensitive mgm1 mutants
(Shepard and Yaffe, 1999).
These results suggest that the GTPase domain of Mgm1p is required for
activity. To test this possibility, we mutated conserved residues in the
GTPase domain among dynamin and dynamin-related GTPases and examined
mitochondrial fusion. Equivalent mutations in dynamin inhibit its GTP binding
and/or hydrolysis (Damke et al.,
2001; Marks et al.,
2001). Using site-directed mutagenesis, we changed three residues
(lysine at residue 223 was changed to alanine, K223A; serine at residue 224
was changed to asparagine, S224N; threonine at residue 244 was changed to
alanine, T244A), creating mgm1K223A,
mgm1S224N, and mgm1T244A alleles,
respectively. Plasmid constructs carrying wild-type and mutant alleles were
integrated into the chromosome in mgm1 dnm1
cells.
We first confirmed that mutant versions of Mgm1p were expressed and
localized to mitochondria. Mitochondria were isolated from cells expressing
wild-type or mutant Mgm1p and analyzed by immune blotting using antibodies to
Mgm1p. Yeast cells contain two species of Mgm1p, a 90- and a 100-kDa form
(Shepard and Yaffe, 1999). We
showed that neither species was expressed in mgm1
dnm1 cells (our unpublished results). As shown in
Figure 3A, similar amounts of
both forms of Mgm1p were found in wild-type and mutant mitochondria. Next, we
examined mitochondria in the different cells using OM45-GFP. We found that
mgm1K223A dnm1, mgm1S224N
dnm1, and mgm1T244A dnm1 mutants
contained tubular mitochondria similar to those seen in mgm1
dnm1 cells, indicating that inactivation of Mgm1p GTPase
disrupts the function of Mgm1p (our unpublished results).
|
We found that a functional GTPase domain of Mgm1p is required for
mitochondrial fusion. Using our mating assay, we examined 50 zygotes for
wild-type and each mutant. As shown in
Figure 3B, all MGM1
dnm1 zygotes fused their mitochondria. In contrast, no
mitochondrial fusion occurred in homozygous zygotes of
mgm1K223A dnm1, mgm1S224N
dnm1, and mgm1T244A dnm1 mutants.
Mgm1p, Fzo1p, and Ugo1p Are Required for Fusion of the Mitochondrial
OM
Our results shown in Figures
2 and
3 indicate that Mgm1p is
required for mitochondrial fusion. However, because mitochondria have two
membranes, an outer and inner membrane, and we used IM-localized YFP and CFP
to assay fusion, it was not possible to determine if Mgm1p was required for OM
fusion, IM fusion, or fusion of both membranes. To ask specifically if OM
fusion is defective in mgm1 and mgm1
dnm1 mutants, we targeted YFP to the mitochondrial OM. In
MAT cells, mitochondria were labeled by an OM-targeted YFP
(TOM72-YFP) carried on pHS72. This construct expresses YFP fused to the first
40 amino acids derived from Tom72p, which includes the transmembrane domain
(Bomer et al., 1996;
Schlossmann et al.,
1996), under the control of the GAL1 promoter. To confirm
that TOM72-YFP is located in the mitochondrial OM, we isolated mitochondria
from cells expressing the YFP fusion. When the mitochondria were treated with
trypsin, TOM72-YFP was completely digested like the OM protein Tom37p (our
unpublished results). The mitochondria in MATa cells were labeled
using an IM-targeted CFP (YTA10-CFP) expressed from pHS70.
In mating between wild-type cells or between dnm1 cells,
CFP and YFP fluorescence in zygotes overlapped in all the mitochondrial
tubules (Figure 4). These
results indicate that the OM of the mitochondria had fused. In contrast, when
mgm1 cells were mated, we found mitochondrial fragments that
contained only CFP or YFP, indicating that these mitochondria did not fuse.
Similarly, in all mgm1 dnm1 zygotes,
mitochondrial tubules contained only one of the two fluorescent markers. These
results demonstrate that mgm1 and mgm1
dnm1 cells are defective in fusion of the mitochondrial
OM.
|
Because Fzo1p and Ugo1p are OM proteins required for mitochondrial fusion,
they have been suggested to mediate fusion of the mitochondrial OM
(Hermann et al.,
1998; Rapaport et
al., 1998; Sesaki and
Jensen, 2001). To test this idea, we examined OM fusion in 50
zygotes formed by mating fzo1 dnm1 cells and
50 zygotes formed by mating ugo1 dnm1 mutants
using TOM72-YFP and YTA10-CFP markers described above. As shown in
Figure 4, both
fzo1 dnm1 and ugo1
dnm1 cells failed to fuse their OMs and contained only YFP or
CFP-labeled mitochondria, similar to mgm1 dnm1
zygotes. Our data indicate Fzo1p and Ugo1p, along with Mgm1p, are required for
fusion of the mitochondrial OM. We note that because OMs do not fuse in cells
lacking Mgm1p, Fzo1p, or Ugo1p, it is not possible to determine if these
proteins are required for IM fusion. Their role in IM fusion therefore awaits
further studies.
Mitochondrial IM Structure Is Altered in mgm1
Cells, but Normal in mgm1 dnm1 Cells
Although our data demonstrate that Mgm1p is required for mitochondrial
fusion, it has been suggested that Mgm1p is involved in formation of IM
structure and that fusion defects seen in mgm1 mutants is a secondary
consequence of the altered IM (Wong et
al., 2000). We examined the mitochondrial morphology of
mgm1 cells by electron microscopy. As shown in
Figure 5a, wild-type cells
contained elongated tubular mitochondria, in which the IM frequently
invaginated into the matrix, forming cristae. In contrast,
mgm1 cells contained small round mitochondria, in which IM
cristae were altered (Figure 5,
c–e). These cristae were longer than those in wild-type
cells and occasionally formed stacks. In addition, the number of cristae in
mgm1 cells appeared to be reduced. The IM structure seen in
mgm1 cells was similar to that seen in wild-type cells lacking
mtDNA (rho0; Figure
5b). Although rho0 cells contained elongated
mitochondrial tubules, the number of cristae in rho0 cells
was less than in wild-type cells carrying mtDNA.
|
In dnm1 cells, mitochondria were more branched and
interconnected, but their IM appeared normal in cristae structure
(Figure 5f). Disruption of
DNM1 restored both mitochondrial tubules and normal IM structure in
mgm1 cells (Figure
5g). In mgm1 dnm1 cells,
mitochondria formed tubular structures and displayed IM cristae
distinguishable from those seen in wild-type cells. Because we showed that
mgm1 dnm1 cells are still defective in
mitochondrial fusion, the fusion defect cannot be due to an alteration of
mitochondrial IM structure. We argue that the fusion defect is the direct
result of a lack of Mgm1p.
To further test the idea that alteration of IM structures affects
mitochondrial fusion, we examined mitochondrial fusion in two mutants, in
which IM structure is highly disorganized, mmm1 and
atp21. In mmm1 cells, the IM cristae collapse
into large membrane sheets, and these sheets often form stacks
(Hobbs et al., 2001).
atp21 cells contain mitochondria whose IM excessively folds
and forms onion-like structures (Paumard
et al., 2002). Using the mating assay, we found that
mitochondria fuse in both mmm1 and atp21
cells. As shown in Figure 6,
zygotes formed between mmm1 cells contained partially
fragmented mitochondria, and these mitochondria carried both YTA10-CFP and
YTA10-YFP, demonstrating that mitochondria fused and mixed their contents. The
mitochondrial shape seen in mmm1 zygotes was different from
that seen in growing mmm1 cells
(Burgess et al., 1994;
Hobbs et al., 2001).
It seems that mitochondria in mmm/ cells change their shape
from large spheres to fragmented tubules during mating.
|
In atp21 zygotes, mitochondria fused normally. We noticed
that although overall patterns of YTA10-CFP and YTA10-YFP signals were very
similar in homozygous zygotes of mmm1 and
atp21 mutants, there were regions of mitochondria where CFP
signals were stronger than YFP, or vice versa. This could be due to slow
diffusion of IM proteins because IM structures in these mutants are remarkably
complex (Hobbs et al.,
2001; Paumard et al.,
2002). Alternatively, it is also possible that mitochondrial
fusion is less efficient in mmm1 and atp21
cells. Nevertheless, these results indicate that aberrant IM structures do not
necessarily block mitochondrial fusion and further support that Mgm1p plays a
direct role in mitochondrial fusion.
Mgm1p Is Associated with Both the Mitochondrial Outer and Inner
Membranes
Although Mgm1p has been localized to mitochondria, its submitochondrial
location is controversial. Shepard and Yaffe
(1999) found Mgm1p in the
mitochondrial OM, exposed to the cytosol. In contrast, Wong et al.
(2000) suggested that Mgm1p is
in the intermembrane space, peripherally associating with the IM. In an
attempt to reconcile these differences, we first examined the association of
Mgm1p with mitochondria. We treated mitochondria with 1.5 M sodium chloride or
0.1 M sodium carbonate (Figure
7A). The Mgm1p protein was extracted from the mitochondria with
sodium carbonate, but not with sodium chloride, like the subunit of the
F1-ATPase, a peripheral membrane protein, confirming the previous
observation (Wong et al.,
2000). Conversely, the integral membrane proteins Om45p
(Yaffe et al., 1989)
and Tim23p (Emtage and Jensen,
1993) remained in the membrane fraction. To ask if Mgm1p is inside
or outside of mitochondria, we treated intact mitochondria using trypsin or
proteinase K. Consistent with the previous observation
(Wong et al., 2000),
we found that Mgm1p was not digested by either of these two proteases
(Figure 7B). As controls, the
OM protein Tom37p (Gratzer et
al., 1995) was completely digested, whereas the IM protein
Aac2p remained intact. When the mitochondrial OM was disrupted by osmotic
shock, trypsin and proteinase K digested Mgm1p, Tom37p, and Aac2p, but not the
matrix protein, Mas2p. Thus, Mgm1p was protected from proteases by the
mitochondrial OM.
|
To ask which membrane Mgm1p is associated with, we prepared membrane
vesicles from mitochondria and separated them into OM and IM fractions on
sucrose gradients. As shown in Figure
7B, the majority of both 100-kDa (
70%) and 90-kDa (100%)
forms of Mgm1p cofractionated with the OM vesicle fraction, along with Om45p.
However, 30% of the 100-kDa form of Mgm1p was also found in the IM
vesicle fraction, along with Cox4p. These results, taken together, indicate
that Mgm1p is peripherally associated with both the mitochondrial outer and
inner membranes in the intermembrane space.
Mgm1p, Ugo1p, and Fzo1p Physically Interact
To test if Mgm1p interacts with Ugo1p and Fzo1p, we asked whether Mgm1p
coimmune precipitated with Ugo1p and Fzo1p from detergent-solubilized
mitochondria. We isolated mitochondria from cells expressing HA-Fzo1p and
solubilized the mitochondria in buffer containing 1% Triton X-100. We then
immune precipitated HA-Fzo1p using antibodies to the HA epitope. The pellet
fraction was analyzed by immune blotting. As shown in
Figure 8A, HA-Fzo1p was found
in the pellet fraction. When we examined the pellet with antibodies to Mgm1p,
we found that the 100-kDa form of Mgm1p coprecipitated along with HA-Fzo1p.
About 5% of the 100-kDa form of Mgm1p was found in the pellet fraction.
Although the 90-kDa form also coprecipitated, the efficiency was lower
(0.1%). Other abundant mitochondrial proteins such as OM45 (an OM
protein) and Tim23p (an IM protein) did not precipitate with HA-Fzo1p. As a
control, we immune precipitated with antibodies to the myc epitope and found
no HA-Fzo1p, Mgm1p, Om45p, or Tim23p in the pellet fractions. We also used
0.5% digitonin to solubilize mitochondria and obtained similar results (our
unpublished results). These results indicate that Mgm1p physically interacts
with Fzo1p and suggest that Mgm1p is a part of the fusion machinery located in
the mitochondrial OM.
|
Mgm1p also associates with Ugo1p. Mitochondria were isolated from cells
expressing myc-Ugo1p, solubilized in Triton X-100-containing buffer. myc-Ugo1p
was then immune precipitated from the detergent-solubilized mitochondria with
antibodies to the myc epitope. Immune blots showed that virtually all
myc-Ugo1p was precipitated (Figure
8B). About 10% of the 100-kDa form of Mgm1p was found in the
pellet fraction. Similar to our precipitation using HA-Fzo1p, only a small
fraction of the 90-kDa form coprecipitated (0.1%). Furthermore, Ugo1p
also interacts with Fzo1p. We found that 3% of Fzo1p coprecipitated along
with Myc-Ugo1p (Figure 8C). In
contrast, Om45p and Tim23p remained in the unbound fraction. As a control, we
incubated mitochondria containing myc-Ugo1p with antibodies to the HA epitope
and found no myc-Ugo1p or Mgm1p precipitated. Thus, Mgm1p, Ugo1p and Fzo1p,
mitochondrial proteins required for outer membrane fusion, physically interact
with each other.
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). In
addition, mgm1 mutants have several striking similarities to
fzo1 and ugo1 mutants
(Jones and Fangman, 1992;
Guan et al., 1993;
Hermann et al., 1998;
Rapaport et al.,
1998; Shepard and Yaffe,
1999; Wong et al.,
2000; Sesaki and Jensen,
2001). mgm1, fzo1, and ugo1 cells all contain
highly fragmented mitochondria and rapidly lose their mtDNA. The fragmentation
of mgm1, fzo1, and ugo1 mitochondria is the result of
ongoing organelle fission in the absence of fusion and can be suppressed in
each mutant by disruption of DNM1, a gene required for the division
of mitochondria (Bleazard et al.,
1999; Sesaki and Jensen,
1999,
2001). Also arguing that Mgm1p
functions in the same pathway as Fzo1p, we find that mgm1
fzo1 dnm1 triple mutants show no additional
mitochondrial defects when compared with fzo1
dnm1 and mgm1 dnm1 double
mutants. The double and triple mutants all contain similarly shaped tubular
mitochondria (H. Sesaki, unpublished observation). In this report we find that
cells lacking Mgm1p are defective in the fusion of their mitochondria and that
the Mgm1 protein physically interacts with both Fzo1p and Ugo1p, arguing that
the Mgm1 protein plays an essential role in mitochondrial fusion.
Mgm1p is related to several, large GTP-binding proteins. The first member
of the family identified was dynamin, which is required for fission of the
clathrin-coated plasma membrane invaginations during the formation of
endocytic vesicles (McNiven,
1998; Schmid et al.,
1998; Sever et al.,
2000b). Supported by in vitro assembly studies, dynamin has been
proposed to form ring-like structures around membrane invaginations
(Takei et al., 1995).
After a GTP-dependent constriction, the dynamin ring is proposed to pinch off
the endocytic vesicle (Sweitzer and
Hinshaw, 1998). Another member of the dynamin family, Dnm1p, is
required for mitochondrial division and is located on the organelle surface
(Bleazard et al.,
1999; Labrousse et
al., 1999; Sesaki and
Jensen, 1999). By analogy to dynamin, Dnm1p may play a direct,
mechanical role in the fission of mitochondria. However, our results show that
another member of the dynamin family, Mgm1p, mediates mitochondrial membrane
fusion, and Gu and Veerma (1996) shows that phragmoplastin, another
dynamin-related protein, plays a role in fusion of Golgi-derived vesicles to
the cell plate during plant cell division. It is possible that the
dynamin-like proteins do not function as "pinchases," but play a
different role in membrane dynamics. For example, similar to the action of
most G-proteins in the cell, dynamin-related proteins may act as regulatory
molecules, recruiting and activating the fission or fusion machinery. Such a
model has been suggested for dynamin (Sever et al.,
1999,
2000a). However, it is
important to note that, at least conceptually, membrane fusion and division
are similar reactions running in opposite directions. Therefore, it may turn
out that analogous molecules mediate both processes.
In matings between cells whose OM was marked by YFP, we find that Mgm1p, Fzo1p, and Ugo1p are all required for fusion of the mitochondrial OM. Moreover, the observation that all three proteins interact suggests that they are part of a fusion machine in the OM. Because Fzo1p and Ugo1p both contain domains facing the cytosol, they are candidates for proteins that mediate the docking and early fusion events between two mitochondria. Mgm1p, which is located inside the OM in the intermembrane space, may organize or activate Fzo1p and Ugo1p and regulate fusion of the OM. Interestingly, both Fzo1p and Mgm1p appear to be GTP-binding proteins. The GTPase domain of Fzo1p faces the cytosol, whereas that of Mgm1p is in the intermembrane space. It is not clear how these two G-proteins coordinate the fusion reaction and further studies are clearly needed to determine the roles of Mgm1p, Ugo1p, and Fzo1p.
Our coimmune precipitation studies demonstrated that Mgm1p, Fzo1p, and
Ugo1p physically interact. Because small fractions of these proteins associate
with each other at steady state, their associations could be transient and/or
weak. Perhaps the small amounts of Mgm1p, Ugo1p, and Fzo1p that interact
represent the small fractions of these proteins actually engaged in
mitochondrial fusion at any given time. Similar short-lived interactions
between SNARE proteins have been observed. About 1% of SNARE proteins assemble
into SNARE complexes and this reflects transient assembly of the SNARE complex
(Grote et al., 2000).
It has been shown that the SNARE complex forms before and/or during fusion and
disassembles immediately after fusion
(Weber et al., 2000).
By analogy, Mgm1p, Fzo1p, and Ugo1p may form protein complexes during
mitochondrial fusion and actively dissociate after fusion. It is tempting to
speculate that all three proteins assemble into the fusion machinery in the
OM. Alternatively, it is also possible that pair-wise interactions of Mgm1p,
Fzo1p, and Ugo1p take place at different steps of the fusion pathway.
Because mgm1, ugo1, and fzo1 mutants are defective in the
fusion of the mitochondrial OM, it is not possible to determine if Mgm1p,
Ugo1p, or Fzo11p plays a role in IM fusion. However, several observations
raise the possibility that the two fusion events may be connected. First,
direct observation of mitochondria fusing after cell mating suggests that the
mitochondrial IM fuses immediately after OM fusion
(Okamoto et al.,
1998; H. Sesaki, unpublished observations). Second, our work with
Mgm1p and previous work with Fzo1p (Fritz
et al., 2001) indicate that the two proteins associate
with both the outer and inner membranes. These two proteins may be located at
contact sites, where the OM and IM are closely positioned. It has been
suggested that OM fusion takes place at contacts sites and coordinates with IM
fusion (Fritz et al.,
2001). Third, the domains of Fzo1p and Ugo1p that face the
intermembrane space are important for their activities
(Fritz et al., 2001;
H. Sesaki, unpublished observations). Nonetheless, whether the OM and IM
contains the same or separate fusion machinery awaits the identification and
analysis of addition fusion components.
In a previous report, Wong et al.
(2000) found that although
mgm1–5 mutants were defective in mitochondrial fusion,
efficient mitochondrial fusion occurred after the mating of two mgm1-5
dnm1 double mutants. They suggested that the fusion defect seen in
mgm1-5 was indirect, resulting instead from IM structural alterations
caused by the lack of Mgm1p. However, using a disruption of the MGM1
open reading frame, we find that both mgm1 and
mgm1 dnm1 mutants are completely defective in
fusion. This inconsistency is not due to different strain backgrounds, because
we see the same defect when MGM1 is disrupted in the W303 strain used
by Wong et al.
(2000). We suggest that the
temperature-sensitive mgm1-5 mutant retains partial Mgm1p activity at
the restrictive temperature. Although the exact role of Mgm1p in mitochondrial
fusion still remained to be determined, the fusion defect seen in
mgm1 and mgm1 dnm1 mutants is
not the consequence of altered IM organization. Disruption of DNM1 in
mgm1 cells restores the IM cristae morphology, yet
mitochondrial fusion still does not occur. Moreover, mmm1 and
atp21 mutants both have drastically altered IM structures
(Hobbs et al., 2001;
Paumard et al.,
2002), but we find that mitochondrial fusion is not blocked in
these mutants.
Because previous studies disagreed about the localization of Mgm1p, we
reexamined its location using isolated mitochondria. Consistent with the
results of Wong et al.
(2000), we found that both
forms of Mgm1p readily extracted from mitochondrial membranes by alkali, and
protease digestion studies indicate that both forms of Mgm1p are located
inside the mitochondrial outer membrane. However, our results differ from Wong
et al. (2000) in that
we find that the majority of Mgm1p is associated with the mitochondrial OM.
Although 30% of the 100-kDa form of Mgm1p comigrated with IM vesicles,
nearly all of the 90-kDa form and 70% of the 100-kDa form cosedimented
with OM vesicles. Perhaps the dual location of Mgm1p explains the apparently
conflicting results seen in different studies.
Mitochondrial fusion appears to be highly conserved from yeast to human,
with orthologues to yeast Fzo1p present in flies
(Hales and Fuller, 1997;
Hwa et al., 2002) and
mammals (Santel and Fuller,
2001; Rojo et al.,
2002). In addition, OPA1 is an apparent human homolog of yeast
Mgm1p and is defective in autosomal dominant optic atrophy, causing an early
childhood visual impairment (Alexander
et al., 2000;
Delettre et al.,
2000). We speculate that OPA1, like its yeast counterpart, is
involved in mitochondrial fusion. Supporting this idea, OPA1 is located in
mitochondria, and cells isolated from patients of the optic atrophy contain
disorganized mitochondria (Delettre et
al., 2000). If OPA1 functions like Mgm1p in mitochondrial
fusion, mitochondria in mammalian cells defective in OPA1would lose their
shape and mtDNA, consistent with the prediction that the optic atrophy results
from defects in respiration and mitochondrial ATP production
(Delettre et al.,
2002). Therefore, further analysis of Mgm1p function will be
important to clarify its role in mitochondrial fusion, but also to better
understand mitochondrial function in general.
|
ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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|
Footnotes |
|---|
Corresponding author. E-mail address:
hsesaki{at}jhmi.edu.
|
REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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